Two estrogen response element sequences near the PCNA gene are not responsible for its estrogen-enhanced expression in MCF7 cells.
BACKGROUND
The proliferating cell nuclear antigen (PCNA) is an essential component of DNA replication, cell cycle regulation, and epigenetic inheritance. High expression of PCNA is associated with poor prognosis in patients with breast cancer. The 5'-region of the PCNA gene contains two computationally-detected estrogen response element (ERE) sequences, one of which is evolutionarily conserved. Both of these sequences are of undocumented cis-regulatory function. We recently demonstrated that estradiol (E2) enhances PCNA mRNA expression in MCF7 breast cancer cells. MCF7 cells proliferate in response to E2.


RESULTS
Here, we demonstrate that E2 rapidly enhanced PCNA mRNA and protein expression in a process that requires ERalpha as well as de novo protein synthesis. One of the two upstream ERE sequences was specifically bound by ERalpha-containing protein complexes, in vitro, in gel shift analysis. Yet, each ERE sequence, when cloned as a single copy, or when engineered as two tandem copies of the ERE-containing sequence, was not capable of activating a luciferase reporter construct in response to E2. In MCF7 cells, neither ERE-containing genomic region demonstrated E2-dependent recruitment of ERalpha by sensitive ChIP-PCR assays.


CONCLUSIONS
We conclude that E2 enhances PCNA gene expression by an indirect process and that computational detection of EREs, even when evolutionarily conserved and when near E2-responsive genes, requires biochemical validation.
Wang
Cheng
C
Department of Gynecology and Obstetrics, Emory University School of Medicine, Atlanta, GA, USA.
Yu
Jie
J
Kallen
Caleb B
CB
D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D019295:Computational Biology; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology*; D004965:Estrogen Antagonists / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology; D000077267:Fulvestrant; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000187:drug effects / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D020218:Response Elements / Q000502:physiology*; D014162:Transfection; D015854:Up-Regulation / Q000187:drug effects
D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger; D000077267:Fulvestrant; D004958:Estradiol